1. Field of the Invention
The present invention relates to a method for detecting the methylation of promoters using HpaII, which is a methylation-sensitive restriction enzyme, and more particularly to a method for detecting the methylation of promoters, which comprises cutting DNA derived from clinical samples or subjects to be diagnosed with restriction enzyme HpaII, amplifying the cut DNA by polymerase chain reaction (PCR) with primers capable of amplifying CpG islands, and determining the presence or absence of the PCR amplification products with a DNA chip for methylation detection.
2. Background of the Related Art
In current clinical practice, the diagnosis of cancer is confirmed by finally performing tissue biopsy after history taking, physical examination and clinical assessment, followed by radiographic testing and endoscopy if cancer is suspected. However, the diagnosis of cancer by the existing clinical practices is possible only when the number of cancer cells is more than a billion, and the diameter of cancer is more than 1 cm. Meanwhile a tumor marker for detecting a substance, which is directly or indirectly produced by cancer in blood is used in cancer screening tests, but it has limitations in accuracy so that it often shows false positive or false negative.
Thus, in order to diagnose and treat cancer at the root, approaches at a gene level need to be performed. Recently, genetic analysis has been actively attempted to diagnose cancer. The simplest typical method is to detect the presence of ABL:BCR fusion genes (genetic characteristic of leukemia) in blood by PCR. Such method has an accuracy of more than 95%, and after the diagnosis and therapy of chronic myelocytic leukemia, this method is being used for the assessment of the result and follow-up study, etc. However, this method has the shortcoming that it can be applied only to some blood cancers.
Furthermore, another method is being attempted, in which the presence of genes expressed by cancer cells is detected by RT-PCR and blotting, thereby diagnosing cancer cells present in blood cells. However, this method has shortcomings in that it can be applied only to some cancers, including prostate cancer and melanoma, and has a high false positive rate. Also, it is difficult to standardize detection and reading in this method, and its utility is also limited (Kopreski, M. S. et al., Clin. Cancer Res., 5:1961, 1999; Miyashiro, I. et al., Clin. Chem., 47:505, 2001).
Recently, genetic testing using a DNA in serum or plasma has been actively attempted. This is a method of detecting a cancer-related gene that is isolated from cancer cells and released into blood and is present in the form of a free DNA in serum. It is found that the concentration of DNA in serum is 5-10 times increased in actual cancer patients as compared to that of normal persons, and such increased DNA is released mostly from cancer cells. The analysis of cancer-specific gene abnormalities, such as the mutation, deletion and functional loss of oncogenes and tumor-suppressor genes, using such DNAs isolated from cancer cells, allows the diagnosis of cancer. There has been an active attempt to diagnose lung cancer, head and neck cancer, breast cancer, colon cancer, and liver cancer, etc., by examining the promoter methylation of mutated K-Ras oncogenes, p53 tumor-suppressor genes and p16 genes in serum, and the labeling and instability of microsatellite (Chen, X. Q. et al., Clin. Cancer Res., 5:2297, 1999; Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedes, M. et al., Cancer Res., 60:892, 2000; Sozzi, G. et al., Clin. Cancer Res., 5:2689, 1999).
Meanwhile, in samples other than blood, the DNA of cancer cells can also be detected. A method is being attempted in which the presence of cancer cells or oncogenes in sputum or bronchoalveolar lavage of lung cancer patients is detected by a gene or antibody test (Palmisano, W. A. et al., Cancer Res., 60:5954, 2000; Sueoka, E. et al., Cancer Res., 59:1404, 1999). Also, other methods of detecting the presence of oncogenes in feces of colon and rectal cancer patients (Ahlquist, D. A. et al., Gastroenterol., 119:1219, 2000) and detecting promoter methylation abnormalities in urine and prostate fluid (Goessl, C. et al., Cancer Res., 60:5941, 2000) are being attempted. However, in order to accurately diagnose cancers that cause a large number of gene abnormalities and show various mutations according to each cancer, a method, by which a large number of genes are simultaneously analyzed in an accurate and automatic manner, is required. However, such a method is not yet established.
Accordingly, methods of diagnosing cancer by the measurement of DNA methylation are being proposed. When the promoter CpG island of a certain gene is over-methylated, the expression of such a gene is silenced. This is interpreted to be a main mechanism in which the function of this gene is lost even when there is no mutation in the protein-coding sequence of the gene in a living body. Also, this is analyzed as a factor by which the function of a number of tumor-suppressor genes in human cancer is lost. Thus, detection of the methylation of the promoter CpG island of tumor-suppressor genes is greatly needed for the study of cancer, and recently, an attempt is actively being conducted in which the promoter methylation by a method, such as methylation-specific PCR (hereinafter, referred to as MSP) or automatic DNA sequencing is examined, thereby enabling the diagnosis and screening of cancer.
For the accurate diagnosis of cancer, it is important to detect not only a mutated gene but also a mechanism by which the mutation of this gene occurs. While previous studies have been conducted by focusing on the mutations of a coding sequence, i.e., micro-changes, such as point mutations, deletions and insertions, or macroscopic chromosomal abnormalities, recently, epigenetic changes are reported to be as important as these mutations, and a typical example of the epigenetic changes is the methylation of promoter CpG islands.
In the genomic DNA of mammal cells, there is the fifth base in addition to A, C, G and T, which is 5-methylcytosine where a methyl group is attached to the fifth carbon of the cytosine ring (5-mC). 5-mC is always attached only to the C of a CG dinucleotide (5′-mCG-3′), which is generally marked CpG. The methylation of this CpG inhibits a repetitive DNA sequence in genomes, such as alu or transposon, from being expressed. Also, this CpG is a site where an epigenetic change in mammal cells occurs most often. The 5-mC of this CpG is naturally deaminated to T, and thus, the CpG in mammal genomes shows only 1% of frequency, which is much lower than a normal frequency (¼×¼=6.25%).
Regions in which CpG is exceptionally integrated are known as CpG islands. The CpG islands refer to sites that are 0.2-3 kb in length, and have a C+G content of more than 50% and a CpG ratio of more than 3.75%. There are about 45,000 CpG islands in the human genome, and they are mostly found in promoter regions regulating the expression of genes. Actually, the CpG islands occur in the promoters of housekeeping genes accounting for about 50% of human genes (Cross, S. H. & Bird, A. P., Curr. Opin. Gene Develop., 5:309, 1995).
Meanwhile, in the somatic cells of normal persons, the CpG islands of such housekeeping gene promoter sites are un-methylated, but imprinted genes and the genes on inactivated X chromosomes are methylated such that they are not expressed during development.
During a cancer-causing process, methylation is found in promoter CpG islands, and the restriction on the corresponding gene expression occurs. Particularly, if methylation occurs in the promoter CpG islands of tumor-suppressor genes that regulate cell cycle or apoptosis, restore DNA, participate in the adhesion of cells and the interaction between cells, and suppress cell invasion and metastasis, it blocks the expression and function of such genes in the same manner as the mutations of a coding sequence, thereby promoting the development and progression of cancer. In addition, partial methylation also occurs in the CpG islands according to aging.
An interesting fact is that, in the case of genes whose mutations are attributed to the development of cancer in congenital cancer but does not occur in acquired cancer, the methylation of promoter CpG islands occurs instead of mutation. Typical examples include the promoter methylation of genes, such as acquired renal cancer VHL (von Hippel Lindau), breast cancer BRCA1, colon cancer MLH1, and stomach cancer E-CAD. In addition, in about half of all cancers, the promoter methylation of p16 or the mutation of Rb occurs, and the remaining cancers show the mutation of p53 or the promoter methylation of p73, p14 and the like.
An important fact is that an epigenetic change caused by this promoter methylation causes a genetic change (i.e., the mutation of a coding sequence), and the development of cancer is progressed by the combination of such genetic and epigenetic changes.
Most of cancers show three common characteristics with respect to CpG, namely, hypermethylation of the promoter CpG islands of tumor-suppressor genes, the hypomethylation of the remaining CpG base sites, and an increase in the activity of methylation enzyme, i.e., DNA cytosine methyltransferase (DNMT) (Singal, R. & Ginder, G. D., Blood, 93:4059, 1999; Robertson, K. & Jones, P. A., Carcinogensis, 21:461, 2000; Malik, K. & Brown, K. W., Brit. J. Cancer, 83:1583, 2000).
When promoter CpG islands are methylated, the reason why the expression of the corresponding genes is blocked is not clearly established, but presumed to be because a methyl CpG-binding protein (MECP) or a methyl CpG-binding domain protein (MBD), and histone deacetylase, bind to methylated cytosine thereby causing a change in the chromatin structure of chromosomes and a change in histone protein.
There is a dispute about whether the methylation of promoter CpG islands directly causes the development of cancer or is a secondary change after the development of cancer. However, it is clear that the promoter methylation of tumor-related genes is an important index to cancer, and thus, can be used in many applications, including the diagnosis and early detection of cancer, the prediction of the risk of the development of cancer, the prognosis of cancer, follow-up examination after treatment, and the prediction of a response to anticancer therapy. Recently, an actual attempt to examine the promoter methylation of tumor-related genes in blood, sputum, saliva, feces or urine and to use the examined results for the diagnosis and treatment of various cancers, is being actively conducted (Esteller, M. et al., Cancer Res., 59:67, 1999; Sanchez-Cespedez, M. et al., Cancer Res., 60:892, 2000; Ahlquist, D. A. et al., Gastroenterol., 119:1219, 2000).
In order to maximize the accuracy of cancer diagnosis using promoter methylation, analyze the development of cancer according to each stage and discriminate a change according to cancer and aging, an examination that can accurately analyze the methylation of all the cytosine bases of promoter CpG islands is required. Currently, a standard method for this examination is a bisulfite genome-sequencing method, in which a sample DNA is treated with sodium bisulfite, and all regions of the CpG islands of a target gene to be examined are amplified by PCR, and then, the base sequence of the amplified regions is analyzed. However, this examination has a problem in that there are limitations on the number of genes or samples that can be examined at a time. Other problems are that automation is difficult, and much time and expense are required.
In Johns Hopkins University, MD Anderson Cancer Center and Medical University of Berlin, etc., studies on the promoter methylation of cancer-related genes are being actively conducted. The fundamental data thus obtained are interchanged through the DNA Methylation Society (DMS) and stored in MethDB, a publicly available DNA methylation database established in 2000 (world wide web address www.methdb.de). Meanwhile, EpiGenX Pharmaceuticals, Inc. is now developing therapeutic agents associated with the methylation of CpG islands, and Epigenomics, Inc. is now conducting studies to apply promoter methylation to cancer diagnosis by examining the promoter methylation using various techniques, such as DNA chips and MALDI-TOF.
Until now, many methods have been attempted to measure the methylation pattern of certain gene promoters. First methods, which comprises cutting each CpG site with a methylation-specific enzyme and then subjecting the cut sites to Southern blot analysis or artificial PCR, have been used (Hatada, I. et al., PNAS, 88:9523, 1991; Liang, G. et al., Methods, 27:150, 2002). Other methods that have been used in the art include methylation-specific PCR (MSP) (Herman, J. G. et al., PNAS, 93:9821, 1996), MethylLight assay (Eads, C. A. et al., Nucleic Acid Res., 28:e42, 2000), and COBRA (Xiong, Z. & Laird, P. W., Nucleic Acid Res., 25:2532, 1997) using DNA modification with sodium bisulfite. However, such technologies are disadvantageous in that they are expensive or difficult to use in clinical applications.
With the recent development of high-throughput analysis technology, assays allowing CpG island methylation to be analyzed at a genome level were developed. Oligonucleotide-based methylation assays using PCR after bisulfite treatment were developed by Adorjan, et al. (Adorjan, P. et al., Nucleic Acid Res., 30:e21, 2002) and Shi et al. (Shi, H. et al., J. Cell Biochem., 88:138, 2003), and also methods such as DMH (Huang, H. T. et al., Human Mol. Genet., 8:459, 1999), CGI (Yan, P. S. et al., Clin. Cancer Res., 6:1432, 2000) and ECISTs (Tsou, J. A. et al., Oncogene, 21:5450, 2002) were developed. Methylation assays using DNA chip include a method comprising modifying the cytosine of genomic DNA into uracil, amplifying the modified DNA, polymerizing the amplification product into oligonucleotide or PNA-oligomer, and hybridizing the polymer in a DNA chip (Korean Patent Laid-open Publication No. 10-2004-0015705).
Such methods have improvements in efficiency and high-throughput analysis over the prior methods, but have problems in that a long time is required and the preparation of samples is complex, thus making it difficult to use such methods in clinical applications. Accordingly, there is now a need for the development of new methodology that can detect methylation quickly in a simple and inexpensive manner and in ever increasing quantities.